Project summary Developing effective bone regeneration therapies is one of the most clinically important long-term goal. Bone loss caused by trauma, neoplasia, reconstructive surgery, congenital defects, or periodontal disease is a major health problem worldwide. Indeed, close to 6 million fractures occur annually in the United States. Of these, 5 to 10% fail to heal properly, due to non-union or delayed union. Clearly, there is a need for safe and effective methods to promote bone regeneration. However, there remain significant challenges in creating bioactive tissues that mimic bone composition and architecture, and can significantly influence cell behavior and performance. We propose to develop novel regenerative strategy to mimic the events occurring during normal bone formation by delivering in situ highly osteoinductive and osteoconductive bone graft. Furthermore, the success of bone healing relies on neovascularization at the injury site. The main causes of graft failure are inner graft necrosis and lack of integration with the circulatory system to provide nutrition and eliminate waste products. Thus, angiogenesis is one of the main unmet clinical needs to ensure successful translation of tissue engineered bone into clinical practice. To achieve precise geometries of the bone structure, 3D bioprinting technology is one of the most appropriate methods for producing 3D multicellular, dynamic and functional bone grafts. Our preliminary data demonstrates that we can successfully synthesize collagen-hydroxyapatite composite bioink and fabricate hybrid scaffold using 3D bioprinting. In addition, we have demonstrated the osteogenic and angiogenic potential of miR-200c and PMIS-miR-200a when delivered to human bone marrow stem cells in vitro, respectively. The final outcome will specifically address drawbacks of local delivery of growth factors to enhance osteogenesis, as well as issues with poor vascularization. The project will initiate with the synthesis and characterization of osteoconductive bioink made by mineralized collagen (Aim 1), followed by the in vitro osteo- angiogenic differentiation of bone marrow stem cells by combinatorial microRNAs gene therapy (Aim 2), and finally assessment of in vivo vascularization and bone formation using 3D bioprinted bone graft (Aim3). The combination of the technologies proposed here will lead to the development of a new functional and effective bone regeneration strategy for oral and maxillofacial reconstruction.